Physiological and biochemical changes associated with acute experimental dehydration in the desert adapted mouse, Peromyscus eremicus

Characterizing traits critical for adaptation to a given environment is an important first step in understanding how phenotypes evolve. How animals adapt to the extreme heat and aridity commonplace to deserts represents is an exceptionally interesting example of these processes, and has been the focus of study for decades. In contrast to those studies, where experiments are conducted on either wild animals or captive animals held in non-desert conditions, the study described here leverages a unique environmental chamber that replicates desert conditions for captive Peromyscus eremicus (cactus mouse). Here we establish baseline values for daily water intake and for serum electrolytes, as well as the response of these variables to experimental dehydration. In brief, P. eremicus’ daily water intake is very low. It’s serum electrolytes are distinct from many previously studied animals, and its response to acute dehydration is profound, though not suggestive of renal impairment, which is atypical of mammals. Summary statement The establishment of baseline values for serum electrolytes and water intake, as well as their response to acute dehydration is critical for characterizing the physiology necessary for desert survival. Conflict of Interest Statement The authors declare no conflict of interest.


Introduction 51
Understanding the evolution of adaptive traits has long been one of the primary 52 goals in evolutionary biology. The study of the relationships between fitness 53 and phenotype, often powered by modern genomic techniques (59), has provided 54 researchers with insight into the mechanistic processes that underlie adaptive 55 phenotypes (15, 28). Systems in which the power of genomics can be combined 56 with an understanding of natural history and physiology are well suited for 57 the study of adaptation (9, 44) especially when researchers have the ability 58 to assay the link between genotype and phenotype in wild animals and then 59 conduct complementary experiments using representative animals in carefully 60 controlled laboratory environments. The study described here, characterizing 61 the physiology and serum biochemistry of Peromyscus eremicus is the first step 62 in a larger study aimed at understanding the genomics architecture of adaptation 63 to desert environments. 64 Desert adaptation has significant ecological, evolutionary, and biomedical 66 significance. In contrast to humans and other mammals, desert rodents can 67 survive in extreme environmental conditions and are resistant to the effects 68 of dehydration. Physiological adaptions to deserts have been characterized in 69 several rodents. Specifically, renal histology has been studied in multiple 70 Heteromyid rodents (3), and the general conclusion is that these desert adapted 71 animals have evolved elongate Loops of Henle (7, 10, 38) that are hypothesized 72 to optimize water conservation. In addition to studies of renal histology, 73 several studies have characterized pulmonary water loss (23, 51), water 74 metabolism (26), and water consumption (12, 34, 35, 41, 46) in desert rodents. 75 While desert animals possess specialized physiology that is efficient with 76 regards to water metabolism and loss, whether or not specialized genomic 77 adaptation exists is an active area of research (32,36,37). 78

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Although the cactus mouse (Peromyscus eremicus) has not been a particular focus 80 for the study of desert adaptation (but see (2, 32), this Cricetid rodent 81 native to the arid regions of the Southwestern United States and Northern 82 Mexico (57) offers a unique opportunity to understand physiological adaptations 83 to deserts.
P. eremicus is a member of a larger genus of animals known 84 colloquially as the "Drosophila of mammals" (9), and Peromyscus species have 85 been the focus of extensive study (25, 33, 52, 54). P. eremicus is a sister 86 species to the non-desert adapted P. californicus (13), and it is closely 87 related to P. crinitus, the canyon mouse, which is another desert adapted 88 rodent native to Southwestern deserts. 89 90 Critical to desert survival is the ability to maintain water balance even when 91 the acute loss of water exceeds dietary water intake (24). Indeed, the mammalian 92 corpus consists of 60% water (30). Far from a static reservoir, proper 93 physiologic function requires water for numerous processes, including nutrient 94 transport (22), signal transduction, pH balance, thermal regulation (42) and 95 the removal of metabolic waste. To accomplish these functions, a nearly constant 96 supply of water is required to replace water loss (30), which occurs mainly 97 via the gastrointestinal and genitourinary systems, and evaporative loss, which 98 is greatly accelerated in extreme heat and aridity (16). Because the body 99 possesses limited reserves, when loss exceeds intake during even a short period 100 of time, dehydration and death can occur. Mammals are exquisitely sensitive to 101 dehydration and possess limited compensatory mechanisms. 102

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Characterizing desert adaptation requires careful and integrative physiological 104 studies, which should include a detailed characterization of water intake, 105 responses to dehydration, and the measurement of blood electrolytes. Indeed, 106 quantifying these metrics is one of the first steps in understanding how animals 107 survive in the extreme heat and aridity of deserts. In particular, the 108 electrolytes chloride and sodium are important markers of dehydration (18). 109 These molecules play essential roles in metabolic and physiological processes, 110 and they are integral to the functionally of a variety of transmembrane 111 transport pumps (11, 29), neurotransmission (62), and maintenance of tonicity 112 (19). Furthermore, hypernatremia causes restlessness, lethargy, muscle weakness, 113 or coma (1). Bicarbonate ion, in contrast, is primarily responsible for aiding 114 in the maintenance of the acid-base balance and is resorbed in the renal tubules 115 (39). Blood urea nitrogen (BUN) is a test that assays the abundance of urea -116 the end-product for metabolism of nitrogen containing compounds. Urea is 117 resorbed in the glomerulus, and renal impairment is often inferred when BUN 118 becomes elevated (8). Importantly, the canonical model of urea resorption is 119 dependent on urine volume, which is markedly diminished in desert rodents, thus 120 limiting the utility of using BUN as an indicator of renal function. Lastly, 121 creatinine, a product of muscle breakdown, whose measured level does not depend 122 on urine volume is used as a measure of renal function (8). ranging from 90F during the daytime to 75F during the night. Relative humidity 145 (RH) ranges from 10% during the day to 25% during the night. Animals are housed 146 in standard lab mouse cages with bedding that has been dehydrated to match 147 desert conditions. They are fed a standard rodent chow, which has also been 148 suggest that no significant differences in any of the physiological measures, 159 and thus, males and females were analyzed as one group. For a subset of animals, 160 water intake was measured, which was accomplished via the use of customized 161 15ml conical tubes, wherein water intake was measured every 24 hours for a 162 minimum of 3 consecutive days (range 3-10 days). Animals selected for the 163 dehydration trial were weighed on a digital scale, housed without water for three days, then re-weighed to determine the change in body mass due to 165 dehydration. At the conclusion of water measurement or after a three-day 166 dehydration animals were sacrificed via isoflurane overdose and decapitation. 167 Immediately after death, a 120uL sample of trunk blood was obtained for serum 168 electrolyte measurement. This was accomplished using an Abaxis Vetscan VS2 We measured the daily water intake for 44 adult cactus mice (24 male, 20 female) 179 for between three and ten consecutive days. Mean water intake was 0.11 mL per 180 day per gram body weight (median=0.11, SD=0.05, min=0.033, max=0.23). We 181 measured levels of serum Sodium, Chloride, Bicarbonate ion, Creatinine, and 182 Blood Urea Nitrogen (BUN) for the same 44 adult mice, thereby establishing 183 normal (baseline) values for P. eremicus ( Figure 1 and Table 1). 184 185 A comparison of mice provided with water ad libitum to mice that exposed to 186 experimental water deprivation for three days revealed that the dehydrated mice 187 lost an average of 23.2% of their body weight (median=23.9%, SD=5.3%, min=12.3%, 188 max=32.3%, n=13 dehydration treatment, 7 males, 6 females).
Despite this 189 substantial weight loss, anecdotally, mice appeared healthy. They were active, 190 eating, and interacting with handlers and other mice, normally. The amount of 191 weight loss did not depend on daily water intake (p=0.63, R 2 = 0.03), though the 192 trend suggests that animals that drink more water lost more weight). Furthermore, 193 body weight did not strongly influence the percent loss of body weight ( Figure  194 2; p=0.68, R 2 = 0.02). 195 In addition to a substantial loss in body weight, dehydration was associated 197 with differences in serum electrolytes (Figure 3; n=13 dehydrated, n=31 198 hydrated). These changes were subtle, but significant using a two-sample t-199 test (p < 0.008 in all cases).